ORIGINAL RESEARCH ARTICLE
published: 18 December 2013
doi: 10.3389/fpls.2013.00508
Kinetic analysis of the interactions between plant
thioredoxin and target proteins
Satoshi Hara 1 and Toru Hisabori 1,2*
1
2
Chemical Resources Laboratory, Tokyo Institute of Technology, Midori-Ku, Yokohama, Japan
Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Tokyo, Japan
Edited by:
Jean-Philippe Reichheld, Centre
National de la Recherche Scientifique,
France
Reviewed by:
Hideki Takahashi, Michigan State
University, USA
David Kramer, Michigan State
University, USA
*Correspondence:
Toru Hisabori, Chemical Resources
Laboratory, Tokyo Institute of
Technology, Nagatsuta 4259-R1-8,
Midori-Ku, Yokohama 226-8503,
Japan
e-mail: [email protected]
Thioredoxin is a critical protein that mediates the transfer of reducing equivalents in vivo
and regulates redox sensitive enzymes in several cases. In addition, thioredoxin provides
reducing equivalents to oxidoreductases such as peroxiredoxin. Through a dithiol–disulfide
exchange reaction, the reduced form of thioredoxin preferentially interacts with the
oxidized forms of targets, which are immediately released after this reaction is complete.
In order to more thoroughly characterize these interactions between thioredoxin and
its target proteins, a mutant version of thioredoxin that lacked the second cysteine
was synthesized and interactions were monitored by surface plasmon resonance. The
binding rates of thioredoxin to its targets were very different depending on the use
of reducing equivalents by the targets: the enzymes whose activity was controlled by
reduction or oxidation of a cysteine pair(s) in the molecule and the enzymes that used
reducing equivalents provided by thioredoxin for their catalysis. In addition, thioredoxin
revealed a stronger preference for an oxidized target. These results explain the reason
for selective association of thioredoxin with oxidized targets for reduction, whereas
immediate dissociation from a reduced target when the dithiol–disulfide exchange reaction
is complete.
Keywords: cysteine, protein–protein interaction, redox regulation, surface plasmon resonance, thioredoxin
INTRODUCTION
Thioredoxin (Trx) is a small, ubiquitous protein that contains a pair of redox-sensitive cysteine residues within its
catalytic domain. This domain comprises the highly conserved
sequence motif Trp-Cys-Gly-Pro-Cys-[Lys/Arg] (Holmgren,
1985; Buchanan, 1991; Balmer et al., 2006). The cysteines allow
Trx to reduce disulfide bonds on target proteins by catalyzing
a dithiol–disulfide exchange reaction, which regulates target
activity in several cases. On the basis of the reduction process
mediated by Trx studies, the cysteine toward the N-terminal
end of the Trx catalytic domain first attacks disulfide bond in a
target protein to form an intermolecular disulfide complex, often
described as a mixed disulfide intermediate complex (Brandes
et al., 1993). Subsequently, the cysteine toward the C-terminal
end of the Trx catalytic domain attacks this intermolecular
disulfide bond, which results in an oxidized form of Trx and a
reduced target.
Trx was first identified as a reducing equivalent donor for the
deoxyribonucleotide reductase in E. coli (Laurent et al., 1964).
In contrast to bacteria and animals, numerous isoforms of Trx
have been identified in plants, although their specific roles are
poorly understood (Balmer et al., 2006). Recent proteomics studies revealed that these Trx isoforms could react with a large
number of candidate target proteins in vitro (Motohashi et al.,
2001; Yano et al., 2001; Balmer et al., 2003; Yamazaki et al., 2004)
and in vivo (Hall et al., 2010). For example, approximately 400
proteins have been described as potential targets of the plant Trx
www.frontiersin.org
system (Montrichard et al., 2009), although only a small portion
of these have been confirmed to be bona fide target proteins by
biochemical analyses.
With regard to the target specificity and the Trx-mediated
reaction process, a number of questions remain unanswered. For
example, the mechanism of disulfide bonds recognition on the
target proteins by Trx and, during the reaction process, the mechanism by which reduced form of Trx react with the oxidized form
of the target protein and release it immediately after the dithiol–
disulfide exchange reaction is completed. The target proteins have
been categorized into two groups on the basis of their mode of
interaction with Trx.
One group is the “switch” type proteins that are predominantly found within the chloroplast such as the chloroplast ATP
synthase γ subunit (McKinney et al., 1979; Nalin and Mccarty,
1984), four Calvin cycle enzymes (glyceraldehyde 3-phosphate
dehydrogenase, fructose 1,6-bis-phsophatase, sedoheptulose 1,7bis-phosphatase, and phosphoribulokinase) (Jacquot et al., 1997),
and malate dehydrogenase (MDH) (Scheibe and Anderson,
1981). The activities of the target proteins in this group are
regulated through the oxidation/reduction of the Trx-targeted
disulfide bond located within the molecule. Once reduced by
Trx, these target proteins no longer require reducing equivalents
provided by Trx.
The second group comprises the “catalytic” types such as
peroxiredoxin (Dietz, 2003) and methionine sulfoxide reductase (MSR) (Tarrago et al., 2009). Because “catalytic” type target
December 2013 | Volume 4 | Article 508 | 1
Hara and Hisabori
enzymes use reducing equivalents provided by Trx as an integral
part of their catalytic process, they must repeatedly interact with
Trx during the catalytic cycle of a target enzyme.
During the dithiol–disulfide exchange reaction, the reduced
form of Trx associates with and reduces the oxidized form of
the target protein, and then the interchange in redox conditions
results in oxidized and reduced forms, respectively. Moreover,
when a disulfide bond on the target protein is reduced by
the reduced form of Trx, the target protein and Trx immediately dissociate for re-reduction of the oxidized form of Trx by
Trx-reductase. This suggests that these proteins must drastically
change their affinity for Trx before and after the dithiol–disulfide
exchange reaction.
Till date, several attempts regarding the interactions between
Trx and the target proteins have been reported. The key residues
on the surface of a Trx-f molecule involved in target specificity were revealed by mutation analyses (Geck et al., 1996;
Geck and Hartman, 2000). The altered S0.5 and Vmax values
for target enzyme activation caused by a Trx mutation indicated the importance of protein–protein interaction in addition to the dithiol–disulfide exchange reaction. In the case
of the interaction between MSR and Trx from E. coli, the
rate constants for the chemical reaction steps were determined
by fluorescence stopped-flow measurements (Antoine et al.,
2003; Olry et al., 2004). The authors concluded that the rate
limiting step for regeneration of the reduced form of MSR
by Trx was the dissociation of oxidized Trx from a reduced
MSR complex.
Considering the Trx reduction reaction, there are two interaction modes between Trx and the target protein that depend on
their redox states. One is the interaction between the reduced
form of Trx and the oxidized form of target protein, and the
other is the interaction between the oxidized form of Trx and
the reduced form of target protein. Clarifying the difference
between these interactions would be helpful for understanding Trx-mediated reduction mechanisms. However, conventional
approaches are difficult to use for revealing the changes in affinity
between Trx and the target proteins, in particular with regard to
the redox states of Trx and its target proteins.
For characterization of these enigmatic but fundamental
phenomena that occur within the Trx catalytic domain, we
used surface plasmon resonance (SPR) to directly determine
the association and dissociation rate constants of Trx to its
target proteins. Arabidopsis thaliana cytosolic MDH and chloroplast peroxiredoxin Q (PrxQ) were used as “switch” and “catalytic” type target model proteins, respectively. Cytosolic MDH
is a homodimeric enzyme that contains a Cys330–Cys330
intermolecular disulfide bond, and reduction of this disulfide
bond by Trx has a strong effect on its activity (Hara et al.,
2006). PrxQ is a chloroplast localized Trx-dependent peroxiredoxin, the activity of which is exhibited efficiently by the
cytosolic Trx rather than the chloroplast Trx (Rouhier et al.,
2004). Moreover, PrxQ is a monomeric protein that contains a Cys45–Cys50 intramolecular disulfide bond. Because
MDH and PrxQ each have a disulfide bond, we selected them
as target model proteins and Trxh as the reductant for this
study.
Frontiers in Plant Science | Plant Physiology
Kinetic analysis of thioredoxin-target interaction
MATERIALS AND METHODS
PROTEINS
Cloning and purification of A. thaliana cytosolic thioredoxin
h1 (Trxh1) (AT3G51030), cytosolic MDH (AT1G04410), and
chloroplast PrxQ (AT3G26060) were performed as described
by Motohashi et al. (2001); Yamazaki et al. (2004); and Hara
et al. (2006), respectively. roGFP1-iL was prepared as described
by Lohman and Remington (2008) with minor modifications.
This protein was used as the redox sensitive GFP derivative
(roGFP) for this study. In brief, roGFP expressed in E. coli strain
BL21(DE3) was purified by anion exchange chromatography
using a DEAE-Toyopearl 650 M and by hydrophobic interaction
chromatography using a Butyl-Toyopearl 650 M.
A Trxh1 mutant with a C43S substitution (Trxh1CS ) and a
Trxh1 mutant with a C40S/C43S substitution (Trxh1SS ) were prepared by the mega-primer method (Sarkar and Sommer, 1990).
These were expressed and purified using the same procedures as
for the wild-type Trxh1 with minor modifications. The Trxh1CS
and Trxh1SS mutants were purified using a solution containing
0.5 mM DTT in all purification procedures to prevent unexpected
disulfide bond formation.
The oxidized MDH was prepared by incubation with 50 μM
CuCl2 for 1 h at 30◦ C. Purified PrxQ and roGFP were obtained as
oxidized forms. To obtain reduced PrxQ, oxidized PrxQ was incubated with 5 mM DTT and then dialyzed against well degassed H
buffer (10 mM HEPES-NaOH, 150 mM NaCl, and 50 μM EDTA,
pH 7.4). The redox states of all proteins were confirmed by the
AMS-labeling method with non-reducing SDS-PAGE (Motohashi
et al., 2001). Protein concentrations were determined using a BCA
assay with BSA as standard.
SPR MEASUREMENTS
SPR measurements were carried out using a Biacore X system
(GE Healthcare, Piscataway, NJ). All experiments were performed
at 25◦ C with the indicated flow rates. All proteins were dialyzed against H buffer before the measurements. Ligand proteins were immobilized on a CM5 sensor chip at a flow rate of
5 μl/min using amino coupling methods. After activation of the
CM5 sensor chip with 0.2 M 1-ethyl-3-(3-dimethylaminopropyl)
carbodiimide and 0.05 M N-hydroxysulfosuccinimide (NHS)
for 7 min, ligand proteins were diluted with 10 mM sodium
acetate (pH 4.5) and immediately injected into the flow cell
(Fc.2) onto the activated CM5 sensor chip using the manual injection mode. When sufficient amounts of ligand proteins were immobilized (equivalent to 800–900 RU signals
on SPR), residual active NHS esters were blocked by injecting 1 M ethanolamine–HCl (pH 8.5) for 7 min. The reference flow cell (Fc.1) was treated in the same way without
ligand proteins.
Experiments were performed after adding 0.005% Surfactant
P20 (GE Healthcare) to H buffer. For repeat measurements, the
surface of the sensor chip was regenerated with 5 mM DTT and
500 mM NaCl in H buffer for 2 min. The association and dissociation phase data (signal from Fc.2–signal from Fc.1) were
used to determine kinetic parameters. A 1:1 binding model was
used to obtain the rate constants for MDH. For PrxQ, data were
fit to a double exponential model that had two individual pairs of
December 2013 | Volume 4 | Article 508 | 2
Hara and Hisabori
Kinetic analysis of thioredoxin-target interaction
association and dissociation rate constants. These analyses were
done using a BIAevalution version 4.1 (GE Healthcare).
RESULTS
DISULFIDE BOND REDUCTION ON roGFP BY Trxh1
We used the following three proteins for this study: MDH, PrxQ,
and roGFP, each of which contained a disulfide bond. We first
determined the rates of disulfide bond reduction in these proteins by wild type Trxh1 (Table 1). Although roGFP has a surface
exposed disulfide bond, it is barely reduced by Trxh1 (Meyer
et al., 2007). We previously reported that the MDH and PrxQ
were reduced by Trx (Motohashi et al., 2001; Hara et al., 2006),
although the reduction rates of these proteins were very different. This difference was probably caused not only by the different
chemical reaction rates of disulfide bond reduction but also by the
differences in protein–protein interactions between Trx and these
target proteins.
Disulfide reduction on roGFP was fluorometrically evaluated
using the excitation ratio of 395 nm/475 nm with emission at
510 nm. Oxidized roGFP (15 μM) was incubated with 0.5 mM
NADPH, 0.5 μM of NADPH-Trx reductase from A. thaliana
(AtNTR), and 1 μM Trxh1 at 25◦ C for 30 min in H buffer. The
excitation spectra were then measured using a FP-8500 spectrofluorometer (JASCO, Tokyo, Japan).
RATE OF DISULFIDE BOND REDUCTION ON MDH MEDIATED BY Trxh1
The extent of disulfide bond reduction was determined from the
proportion of reduced forms in total MDH proteins. To reduce
Trxh1, 0.5 mM NADPH, 0.5 μM AtNTR, and 0.2–5 μM Trxh1
were incubated at 25◦ C for 5 min in H buffer in advance. The
reaction was initiated by adding 1 μM oxidized MDH to this
mixture, and at the indicated times, proteins in the aliquot were
precipitated by adding 5% TCA (w/v). SDS-sample buffer containing 10 mM N-ethylmaleimide (NEM) was then added to these
precipitants and proteins were separated by non-reducing SDSPAGE. The band intensities (Ired and Iox for reduced and oxidized
proteins, respectively) were determined using a Scion Image software and the reduced proportion was calculated by the following
equation: Reduced proportion (%) = Ired /(Ired + Iox ) × 100%.
DISULFIDE BOND REDUCTION RATE ON PrxQ BY Trxh1
The rate of disulfide bond reduction on PrxQ was estimated from
the H2 O2 reduction activity of PrxQ. This activity was determined using a coupling assay system that contained AtNTR and
Trx. The decrease in absorbance at 340 nm due to NADPH oxidation was monitored (Motohashi et al., 2001). The reaction was
initiated by adding 3 μM Trx to a reaction mixture that contained
0.5 mM NADPH, 1 μM AtNTR, 0.5 μM PrxQ, and 0.5 mM H2 O2
in H buffer.
GEL FILTRATION CHROMATOGRAPHY
Gel filtration chromatography analysis was performed using a
TSK-G2000SWXL (Tosoh, Tokyo), which had been equilibrated
with H buffer. Eluted proteins were monitored at 280 nm.
REDUCTION OF DISULFIDE BOND CONTAINING PROTEINS BY WILD
TYPE Trxh1
TARGET PREFERENCES REVEALED BY SPR MEASUREMENTS
We first attempted SPR measurement using a combination of wild
type Trxh1 as a ligand and oxidized MDH as the analyte; however, no SPR signals could be detected using this combination.
Thus, we used the Trxh1CS mutant as the ligand in subsequent
experiments. The Trxh1CS lacks the second cysteine in its catalytic
domain and cannot catalyze the complete exchange of disulfide
bond pairs with the target proteins. Thus, this appeared to be a
good experimental model protein to capture a snapshot of the
dithiol–disulfide exchange reaction mediated by Trx. Although
this mutant cannot be used to imitate the actual affinity due to
the formation of a mixed disulfide bond, this mutant exhibited
association and dissociation activity in our SPR measurements.
Therefore, we describe the data obtained in this study as apparent
affinities for Trx and the target proteins.
Using the Trxh1CS , we attempted to observe the dissociations
and associations between Trx and three proteins, roGFP, PrxQ,
and MDH, as SPR signals. These proteins have redox-sensitive
disulfide bonds in their oxidized states. Thus, the resulting SPR
signals should have been due to the formation of mixed disulfide
intermediate complexes. When these proteins were injected into
the Trxh1CS immobilized on a CM5 sensor chip for SPR measurements, the sensorgrams for the association phase were very
different (Figure 1). When roGFP with a surface-based Trx irreducible disulfide bond was used as the analyte, the SPR signals
Table 1 | Rate constants of target proteins interacting with Trxh1.
Method
Rates
kon (M−1 s−1 )
SPRa
koff
Reduction reaction
a Association
b Reduction
(s−1 )
Reduction rate (s−1 )
roGFP
MDH
PrxQ
Rapid
Slow
N.D.
247
7.97 × 104
765
N.D.
3.01 × 10−4
0.62
5.62 × 10−4
N.D.
6.5 × 10−3b
0.67c
and dissociation rate constants were determined by using Trxh1CS mutant.
rate of MDH was determined from the reduction extent of MDH by wild type Trxh1 measured at the various reaction periods (for detail, see Materials
and Methods).
c Reduction
rate of PrxQ was determined from the amount of NADPH consumption in the presence of AtNTR, wild type Trxh1, PrxQ and hydrogen peroxide. The
amounts of PrxQ was compensated to be a rate limiting step (for detail, see Materials and Methods).
www.frontiersin.org
December 2013 | Volume 4 | Article 508 | 3
Hara and Hisabori
Kinetic analysis of thioredoxin-target interaction
FIGURE 1 | Associations of model proteins with the Trxh1CS . PrxQ,
MDH, or roGFP at 5 μM were injected onto the Trxh1CS immobilized on a
CM5 sensor chip. Experiments were performed at a flow rate of 30 μl/min
for PrxQ and 10 μl/min for MDH and roGFP.
were barely detectable. This suggested that the Trxh1CS did not
associate non-specifically with this protein, even if the protein
contains disulfide bond(s) on its surface. In contrast, PrxQ and
MDH provided more significant SPR signals. These sensorgrams
clearly revealed that the binding rate of PrxQ to the Trxh1CS
was much faster than that of MDH. These results were in accordance with the significant differences in their reduction rates of
the disulfide bonds located on MDH (6.5 × 10−3 s−1 ) and PrxQ
(0.67 s−1 ) by the wild type Trxh1 (Table 1).
KINETIC ANALYSIS OF THE INTERACTION BETWEEN MDH AND Trx
To thoroughly investigate the interaction between the MDH and
Trxh1CS , we determined the rate constants for association and
dissociation with the Trxh1CS using SPR (Figure 2A). These data
were fit to a 1:1 binding model, from which we obtained the
association rate constant, kon = 2.5 × 102 M−1 s−1 , and dissociation rate constant, koff = 3.0 × 10−4 s−1 (Table 1). MDH
dissociated from the immobilized Trxh1CS during these measurements, although the increase in the SPR signals that were observed
indicated the formation of an irreversible disulfide bond.
We then used gel filtration chromatography to determine the
cause for MDH dissociation from the Trxh1CS mutant. To form
a mixed disulfide intermediate complex between the Trxh1CS and
MDH, 44 μM of the oxidized form of MDH was incubated with
160 μM Trxh1CS for 30 min at 25◦ C. This protein mixture was
then applied to the TSK-G2000SWXL column and the desired
peak with a retention time of 13.5 min that contained the mixed
disulfide intermediate complexes was fractionated (orange colored portion in Figure 2B). The thiol alkylation reagent NEM was
immediately added to this collected fraction and chromatography
was repeated (Figure 2B).
When NEM was added to this fraction, the mixed disulfide intermediate complexes were maintained during the second
round of chromatography and the protein peak on the second
chromatography run was observed at the same retention time.
In contrast, the intermediate protein complexes readily dissociated and MDH dimers were observed when the second round
of chromatography was performed without NEM treatment in
advance. These results clearly indicated that the dissociation of the
Frontiers in Plant Science | Plant Physiology
FIGURE 2 | Interaction between the oxidized form of the MDH and
Trxh1CS proteins. (A) To investigate the association and dissociation
phases, the indicated concentrations of MDH were injected for 600 s,
followed by injection by buffer for additional 600 s (left panel) and the SPR
signals were recorded. Fitted curves are indicated by the black lines on the
SPR sensorgram. The association and dissociation phases are indicated by
bars on the sensorgram. The arrow indicates the time when 5 mM DTT was
injected (right panel). (B) Mixed disulfide intermediate complexes formed
by the Trxh1CS and oxidized MDH were separated by gel filtration
chromatography (TSK-G2000SWXL ) (black trace). The intermediate
complexes (orange colored portion) were fractionated and then applied to
the same column chromatography after incubation for the indicated period
in the presence (blue trace) or the absence (red trace) of 10 mM NEM.
mixed disulfide intermediate proteins occurred following nucleophilic attack on the disulfide bond by the thiol residue on the
target protein, presumably observed as dissociation on the SPR
sensorgram.
KINETIC ANALYSIS OF THE INTERACTION BETWEEN PrxQ AND Trx
The sensorgram for the interaction between the Trxh1CS and
PrxQ is presented in Figure 3A. Both association and dissociation phases revealed a biphasic interaction. Thus, these data were
fit to a double-exponential equation, from which we obtained
two rate constant pairs (Table 1). To identify the protein complexes involved in these observed biphasic reactions, 50 μM
December 2013 | Volume 4 | Article 508 | 4
Hara and Hisabori
Kinetic analysis of thioredoxin-target interaction
revealed two binding phases, faster and slower, we concluded
that the faster phase most possibly corresponded to the formation of the mixed disulfide intermediate complex and the latter
corresponded to the formation of the 45 kDa protein complex
(Figure 3C).
We then used gel filtration chromatography to determine whether the rapid dissociation rate (koff−rapid = 0.62 s−1 )
observed with SPR measurements corresponded to the release of
the target protein from the mixed disulfide intermediate complex.
For this purpose, 50 μM Trxh1CS and 50 μM PrxQ were incubated for 3 min at 25◦ C to prepare mixed disulfide intermediate
complexes, and then this protein mixture was treated with 10 mM
NEM. Proteins were then loaded onto the TSK-G2000SWXL .
Mixed disulfide intermediate complexes were only observed when
prior treatment with NEM was used (Figures 3D,E). This indicated that mixed disulfide intermediate complexes comprising the
Trxh1CS and PrxQ would completely dissociate into monomeric
proteins during gel filtration chromatography, similar to the
MDH/Trxh1CS complexes when the second cysteines on PrxQ
were functional.
These data suggested that the rate constants obtained for the
rapid phase presented in Table 1 corresponded to the formation
and reverse dissociation reactions of mixed disulfide intermediate
complexes.
REDOX STATE OF THE TARGET PROTEINS IS REQUIRED FOR EFFICIENT
INTERACTION
FIGURE 3 | Interaction between the oxidized form of the PrxQ and
Trxh1CS proteins. (A) Same experiment as presented in Figure 2A, except
using PrxQ as the analyte. (B) Trxh1CS (50 μM) and PrxQ (50 μM) were
incubated for the periods indicated. The reaction was then quenched with
5% TCA (w/v). Proteins were separated by non-reducing SDS-PAGE. (C)
Band intensities of the mixed disulfide intermediate complexes
(intermediate), 45 kDa complexes, and the Trxh1CS dimers (Trx dimer)
obtained from SDS-PAGE presented in panel (B) were determined using a
Scion image software and plotted. a.u.; arbitrary units. (D) Trxh1CS (50 μM)
and oxidized PrxQ (50 μM) were incubated for 3 min at room temperature,
and the complexes that formed were then incubated with 10 mM NEM.
The protein mixture was then loaded onto G2000SWXL and separated. (E)
Same experiment as in (D) except for incubation in the absence of NEM.
Trxh1CS and 50 μM PrxQ were incubated for the periods indicated (Figures 3B,C). Mixed disulfide intermediate complex formation reactions were then quenched by adding 5% TCA (w/v)
and proteins were separated by non-reducing SDS-PAGE.
From N-terminal amino acid sequence analysis, we confirmed
that the two bands labeled “45 kDa” and “intermediate” were
composed of the Trxh1CS and PrxQ through intermolecular disulfide bonds (Figure 3B). Although the stoichiometry for these proteins in this complex remains to be determined, the 45 kDa protein could be composed of one Trxh1CS and two PrxQ molecules
linked through the intermolecular disulfide bonds. The apparent
formation rate for the one-to-one mixed disulfide intermediate
complex between the Trxh1CS and PrxQ was sufficiently fast and
formation of this complex reached a plateau in less than 15 s.
The Trxh1CS dimer formation and 45 kDa band formation
occurred after this mixed disulfide intermediate complex was
formed (Figure 3C). Because the SPR sensorgram additionally
www.frontiersin.org
Trx obviously recognizes the redox state of the target proteins
in vivo to efficiently reduce them. We aimed to determine whether
the redox state of the target protein affects the affinity between
the target protein and Trx. We measured the binding of MDH
and PrxQ as analytes to the Trxh1CS by SPR. When the reduced
form of MDH was injected onto the Trxh1CS immobilized on a
CM5 sensor chip, no SPR signals were detected, although sufficient binding could be detected when oxidized MDH was used
(Figure 4A).
In contrast, the reduced form of PrxQ could partially associate with the Trxh1CS , although the sensorgram clearly revealed a
quantitative difference as compared to the oxidized form of PrxQ
(Figure 4B). In fact, the reduced form of PrxQ exhibited immediate association and dissociation with the Trxh1CS in a manner
similar to that of the oxidized form of PrxQ. However, the SPR
signals with the reduced form of PrxQ disappeared immediately
when buffer was injected onto the sensor chip, which indicated
that the affinity between the Trxh1CS and the reduced form of
PrxQ was very weak.
SIGNIFICANCE OF PROTEIN–PROTEIN INTERACTIONS
Finally, to investigate the contribution of the surface properties of
Trx for its interaction with the target proteins, we examined the
association of Trxh1SS with PrxQ by substitution of both the cysteines with serine. As presented in Figure 5A, the Trxh1SS interacted with the oxidized form of PrxQ. This interaction between
the Trxh1SS and the oxidized form of PrxQ was not affected by
alkylation of the additional cysteine at position 11 of the immobilized Trxh1SS using 10 mM NEM (Figure 5B). This suggested that
December 2013 | Volume 4 | Article 508 | 5
Hara and Hisabori
FIGURE 4 | Changes in affinity based on the redox states of the target
proteins. (A) Reduced or oxidized MDH (5 μM) was injected onto the
Trxh1CS immobilized on a sensor chip and SPR signals were recorded. (B)
Reduced or oxidized PrxQ (5 μM) was injected onto the Trxh1CS
immobilized on a sensor chip and SPR signals were recorded.
the observed binding of the oxidized form of PrxQ to the immobilized Trxh1SS was due to a protein–protein interaction without
intermolecular disulfide bond formation. The observed binding
rate was slower than that for the Trxh1CS .
DISCUSSION
Several studies attempted to evaluate the affinities of Trx to its
target proteins or the reduction efficiencies of the target proteins by determining Km or kcat /Km values obtained from the
results of complete reduction of the target proteins using Trx
(Collin et al., 2003; Perez-Perez et al., 2009; Maeda et al., 2010).
However, a rather enigmatic behavior of Trx remained unresolved; the reduced form of Trx preferentially interacts with the
oxidized form of the target protein and the oxidized form of Trx
must release the reduced form of the target in order to ensure
an efficient reduction cycle. To address this issue, we applied
the direct binding measurements to detect the associations and
dissociations of Trx with its target proteins.
For this purpose, MDH, PrxQ, and roGFP were selected as
model target proteins. Cytosolic MDH is reportedly a target protein for Trxh1 (Hara et al., 2006) whose activity is regulated by
its reduction/oxidation. Therefore, we defined this type of target protein as a “switch” type target for Trx. In contrast, PrxQ
uses reducing equivalents provided by Trx for catalysis; thus, we
defined it as a “catalytic” type target. Although PrxQ is localized in the chloroplasts of higher plants, cytosolic Trx can be an
efficient reductant for this peroxiredoxin in vitro, as previously
described (Rouhier et al., 2004). In addition, the reduction rate
of PrxQ by Trxh1 (0.67 s−1 ) obtained in this study was comparable to or higher than the reduction rate of PrxQ reduced by
Frontiers in Plant Science | Plant Physiology
Kinetic analysis of thioredoxin-target interaction
FIGURE 5 | Interactions between the Trxh1SS and the reduced and
oxidized forms of PrxQ. (A) Reduced or oxidized PrxQ (5 μM) was injected
onto the Trxh1SS immobilized on a sensor chip and SPR signals were
recorded. (B) The oxidized form of PrxQ (2 μM) was injected onto Trxh1SS
immobilized on a CM5 sensor chip before (gray line) and after (black line)
thiol alkylation. Thiol alkylation was accomplished by injecting 10 mM NEM
for 10 min onto the sensor chip.
chloroplast type Trx (Collin et al., 2004; Perez-Perez et al., 2009).
In this study, we selected a combination of PrxQ and Trxh1 for
our binding study because PrxQ has only one pair of cysteines
in its monomeric molecule and its reaction mode is the simplest
among peroxiredoxins.
When the wild type Trxh1 was used as a ligand, no SPR signals
were detected with MDH as the analyte. Based on the reaction
mechanism of Trx-mediated reduction of a target disulfide bond,
the target protein should be immediately released from Trx when
the reduction reaction is complete. This implies that the number
of bound target proteins on immobilized Trx should be statistically limited by the duration of the reaction period from the
formation of a mixed disulfide bond to its reduction. The time
during which the target protein remains on immobilized Trx
may be too short to allow for detecting the bound molecules on
the gold surface of an SPR sensor chip. In addition, when the
reduction reaction is completed by the dithiol–disulfide exchange
reaction, immobilized Trxh1 adopts its oxidized form and can no
longer reduce the target protein because there is no way to simultaneously reduce immobilized Trxh1 alone. Thus, a direct analysis
of the binding affinity of Trx to its target protein is difficult.
Therefore, we used the immobilized Trxh1CS , which had been
used for Trx affinity chromatography (Yamazaki et al., 2004), as
the ligand in our SPR measurements. Because the Trxh1CS could
efficiently capture the target protein molecules through intermolecular disulfide bond formation, we expected that this mutant
Trx could be used for observing the formation of a mixed disulfide
intermediate complex, which is the initial contact of Trx and its
target protein during the entirety of a reduction reaction. In contrast, a Trxh1SC mutant, a Trxh1 mutant with a C40S substitution,
December 2013 | Volume 4 | Article 508 | 6
Hara and Hisabori
was not used for these measurements because this mutant could
not capture any specific target proteins when used for Trx affinity
chromatography (Yamazaki et al., 2004).
As presented in Figure 1, SPR signals for the association of
the analyte protein with the ligand were successfully obtained
by using the Trxh1CS as the ligand. In contrast, the Trxh1CS did
not associate with roGFP, which contains a disulfide bond that
cannot be reduced by Trx. This indicated that the immobilized
Trxh1CS maintained specificity for the target proteins. In addition, the remarkable differences between the binding time courses
for MDH and PrxQ (Figure 1) suggested that the method applied
in this study was useful for our purposes.
Because the fast dissociation rate of the analyte from the
mixed disulfide intermediate complex by the reverse reaction
(koff−rapid = 0.62 s−1 ) appear to pose difficulty in accurate determination of the association rate constant when performing SPR
analysis using PrxQ, we concluded that the association rate
constant for the formation of the mixed disulfide intermediate
complex was in the order of 104 M−1 s−1 . In addition, the association rate constant for PrxQ was higher than that for MDH by
two orders of magnitude. These results were comparable to the
reported differences in the reduction rates of PrxQ and MDH
by the wild type Trxh1; 0.67 s−1 and 6.5 × 10−3 s−1 , respectively
(Table 1).
Why did Trx (Trxh1CS ) that mimics the reduced form of
Trx interact with the oxidized form of MDH but not with the
reduced form as presented in Figure 4A? A significant conformational change during the transition between the oxidized form
and the reduced form of an MDH dimer has been suggested
by size exclusion chromatography and analytical ultracentrifugation (Hara et al., 2006). This large conformational change must
be critical for forming an intermolecular disulfide bond between
Cys330–Cys330. In addition, the structure or the molecular surface of MDH for its interaction with Trx may only be exposed
in its oxidized form but not its reduced form because on the
basis of its reported crystal structure, the two Cys330 residues are
located at the opposite sides in an MDH dimer molecule. This
may be the reason for interaction of the Trxh1CS with the oxidized form of MDH but not with the reduced form as presented in
Figure 4A. In addition, a redox-dependent large conformational
change has been reported for the other switch type target protein, HSP33. For HSP33, which is known to be a redox-dependent
chaperone that contains four redox responsive cysteines, the
domain that contains Trx-targeted disulfide bonds is largely
unfolded in the oxidized form, although the reduced form of
HSP33 is completely folded and coordinates zinc in this domain
(Ilbert et al., 2007).
In contrast, the Trxh1CS could bind to the reduced form of
PrxQ as presented in Figure 4B. On the basis of the reported
structures of the reduced and oxidized forms of PrxQ from
Aeropyrum pernix (ApPrxQ), a redox-dependent conformational
change was clearly observed at the region that contained two
active cysteines (Perkins et al., 2012). In contrast, molecular modeling for BCP1, the homolog of PrxQ in Sulfolobus solfataricus,
and its reductase, SsPDO (Limauro et al., 2010), revealed that the
stable regions such as the end of β3 and α3 were involved in the
molecular interaction, although these regions do not change their
www.frontiersin.org
Kinetic analysis of thioredoxin-target interaction
conformations in ApPrxQ in a redox-dependent manner. These
reports suggest that a certain stable region on a PrxQ protein can
interact with its partner protein irrespective of their redox states.
The reduced form of PrxQ may have a similar interaction region
on its molecular surface, and it may have caused the relatively
weak association between the Trxh1CS and the reduced form of
PrxQ in our SPR experiments.
In this study, we conclusively confirmed that the redox state of
the target protein was the significant determinant for its affinity to
Trx. As presented in Figure 4, MDH and PrxQ altered their affinities for Trxh1. As previously noted, the Trxh1CS cannot associate
with the reduced form of MDH. The amount of the reduced form
of PrxQ that associated with Trx was definitely less than that of its
oxidized form. These results strongly suggest that the interaction
between Trx and an oxidized target protein exhibits a higher affinity than that between Trx and the reduced form, a prerequisite for
efficient association and subsequent dissociation.
In addition, the Trxh1SS exhibited a preference for the oxidized
target protein, even though its active domain lacks two cysteines
(Figure 5). This indicates that the protein–protein interaction
defined by the molecular surfaces of Trx and its target proteins is
an important determinant for the redox-dependent selectivity of
Trx, in addition to the cysteines in the catalytic domain of Trx and
the rate of formation of the mixed disulfide intermediate complex. Similarly, the interaction between a Trx mutant that lacked
two cysteines and its target ATP synthase complex was previously
reported (Stumpp et al., 1999).
Considering together, these data reveal that the efficient reduction of the target proteins by Trx is accomplished by a remarkable
change in the affinity between Trx and its target protein before
and after the dithiol–disulfide exchange reaction. This selectivity
is defined by these protein–protein interactions. The key molecular determinants of Trx that confer these interesting properties
need to be elucidated by structural analysis of the Trx–target
protein co-complexes.
REFERENCES
Antoine, M., Boschi-Muller, S., and Branlant, G. (2003). Kinetic characterization of
the chemical steps involved in the catalytic mechanism of methionine sulfoxide
reductase A from Neisseria meningitidis. J. Biol. Chem. 278, 45352–45357. doi:
10.1074/jbc.M307471200
Balmer, Y., Koller, A., Del Val, G., Manieri, W., Schurmann, P., and Buchanan,
B. B. (2003). Proteomics gives insight into the regulatory function of
chloroplast thioredoxins. Proc. Natl. Acad. Sci. U.S.A. 100, 370–375. doi:
10.1073/pnas.232703799
Balmer, Y., Vensel, W. H., Hurkman, W. J., and Buchanan, B. B. (2006). Thioredoxin
target proteins in chloroplast thylakoid membranes. Antioxid. Redox Signal. 8,
1829–1834. doi: 10.1089/ars.2006.8.1829
Brandes, H. K., Larimer, F. W., Geck, M. K., Stringer, C. D., Schurmann, P., and
Hartman, F. C. (1993). Direct identification of the primary nucleophile of
thioredoxin f. J. Biol. Chem. 268, 18411–18414.
Buchanan, B. B. (1991). Regulation of CO2 assimilation in oxygenic photosynthesis: the ferredoxin/thioredoxin system. Perspective on its discovery,
present status, and future development. Arch. Biochem. Biophys. 288, 1–9. doi:
10.1016/0003-9861(91)90157-E
Collin, V., Issakidis-Bourguet, E., Marchand, C., Hirasawa, M., Lancelin, J. M.,
Knaff, D. B., et al. (2003). The Arabidopsis plastidial thioredoxins: new functions
and new insights into specificity. J. Biol. Chem. 278, 23747–23752. doi:
10.1074/jbc.M302077200
Collin, V., Lamkemeyer, P., Miginiac-Maslow, M., Hirasawa, M., Knaff, D. B.,
Dietz, K. J., et al. (2004). Characterization of plastidial thioredoxins from
December 2013 | Volume 4 | Article 508 | 7
Hara and Hisabori
Arabidopsis belonging to the new y-type. Plant Physiol. 136, 4088–4095. doi:
10.1104/pp.104.052233
Dietz, K. J. (2003). Plant peroxiredoxins. Annu. Rev. Plant Physiol. Plant Mol. Biol.
54, 93–107. doi: 10.1146/annurev.arplant.54.031902.134934
Geck, M. K., and Hartman, F. C. (2000). Kinetic and mutational analyses of
the regulation of phosphoribulokinase by thioredoxins. J. Biol. Chem. 275,
18034–18039. doi: 10.1074/jbc.M001936200
Geck, M. K., Larimer, F. W., and Hartman, F. C. (1996). Identification of residues
of spinach thioredoxin f that influence interactions with target enzymes. J. Biol.
Chem. 271, 24736–24740. doi: 10.1074/jbc.271.40.24736
Hall, M., Mata-Cabana, A., Akerlund, H. E., Florencio, F. J., Schroder, W. P.,
Lindahl, M., et al. (2010). Thioredoxin targets of the plant chloroplast lumen
and their implications for plastid function. Proteomics 10, 987–1001. doi:
10.1002/pmic.200900654
Hara, S., Motohashi, K., Arisaka, F., Romano, P. G., Hosoya-Matsuda, N., Kikuchi,
N., et al. (2006). Thioredoxin-h1 reduces and reactivates the oxidized cytosolic
malate dehydrogenase dimer in higher plants. J. Biol. Chem. 281, 32065–32071.
doi: 10.1074/jbc.M605784200
Holmgren, A. (1985). Thioredoxin. Annu. Rev. Biochem. 54, 237–271. doi:
10.1146/annurev.bi.54.070185.001321
Ilbert, M., Horst, J., Ahrens, S., Winter, J., Graf, P. C., Lilie, H., et al. (2007). The
redox-switch domain of Hsp33 functions as dual stress sensor. Nat. Struct. Mol.
Biol. 14, 556–563. doi: 10.1038/nsmb1244
Jacquot, J. P., Lancelin, J. M., and Meyer, Y. (1997). Thioredoxins: structure
and function in plant cells. New Phytol. 136, 543–570. doi: 10.1046/j.14698137.1997.00784.x
Laurent, T. C., Moore, E. C., and Reichard, P. (1964). Enzymatic synthesis of
deoxyribonucleotides. IV. Isolation and characterization of thioredoxin, the
hydrogen donor from Escherichia coli B. J. Biol. Chem. 239, 3436–3444.
Limauro, D., D’Ambrosio, K., Langella, E., de Simone, G., Galdi, I., Pedone, C., et al.
(2010). Exploring the catalytic mechanism of the first dimeric Bcp: functional,
structural and docking analyses of Bcp4 from Sulfolobus solfataricus. Biochimie
92, 1435–1444. doi: 10.1016/j.biochi.2010.07.006
Lohman, J. R., and Remington, S. J. (2008). Development of a family of
redox-sensitive green fluorescent protein indicators for use in relatively oxidizing subcellular environments. Biochemistry 47, 8678–8688. doi: 10.1021/
bi800498g
Maeda, K., Hagglund, P., Bjornberg, O., Winther, J. R., and Svensson, B.
(2010). Kinetic and thermodynamic properties of two barley thioredoxin
h isozymes, HvTrxh1 and HvTrxh2. FEBS Lett. 584, 3376–3380. doi:
10.1016/j.febslet.2010.06.028
McKinney, D. W., Buchanan, B. B., and Wolosiuk, R. A. (1979). Association of
a thioredoxin-like protein with chloroplast coupling factor (CF1). Biochem.
Biophys. Res. Commun. 86, 1178–1184. doi: 10.1016/0006-291X(79)90241-9
Meyer, A. J., Brach, T., Marty, L., Kreye, S., Rouhier, N., Jacquot, J. P., et al. (2007).
Redox-sensitive GFP in Arabidopsis thaliana is a quantitative biosensor for the
redox potential of the cellular glutathione redox buffer. Plant J. 52, 973–986. doi:
10.1111/j.1365-313X.2007.03280.x
Montrichard, F., Alkhalfioui, F., Yano, H., Vensel, W. H., Hurkman, W. J., and
Buchanan, B. B. (2009). Thioredoxin targets in plants: the first 30 years.
J. Proteomics 72, 452–474. doi: 10.1016/j.jprot.2008.12.002
Motohashi, K., Kondoh, A., Stumpp, M. T., and Hisabori, T. (2001).
Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc.
Natl. Acad. Sci. U.S.A. 98, 11224–11229. doi: 10.1073/pnas.191282098
Frontiers in Plant Science | Plant Physiology
Kinetic analysis of thioredoxin-target interaction
Nalin, C. M., and Mccarty, R. E. (1984). Role of a disulfide bond in the γ subunit
in activation of the ATPase of chloroplast coupling factor 1. J. Biol. Chem. 259,
7275–7280.
Olry, A., Boschi-Muller, S., and Branlant, G. (2004). Kinetic characterization of
the catalytic mechanism of methionine sulfoxide reductase B from Neisseria
meningitidis. Biochemistry 43, 11616–11622. doi: 10.1021/bi049306z
Perez-Perez, M. E., Martin-Figueroa, E., and Florencio, F. J. (2009). Photosynthetic
regulation of the cyanobacterium Synechocystis sp. PCC 6803 thioredoxin system and functional analysis of TrxB (Trx x) and TrxQ (Trx y) thioredoxins. Mol.
Plant 2, 270–283. doi: 10.1093/mp/ssn070
Perkins, A., Gretes, M. C., Nelson, K. J., Poole, L. B., and Karplus, P. A. (2012).
Mapping the active site helix-to-strand conversion of CxxxxC peroxiredoxin Q
enzymes. Biochemistry 51, 7638–7650. doi: 10.1021/bi301017s
Rouhier, N., Gelhaye, E., Gualberto, J. M., Jordy, M. N., de Fay, E., Hirasawa,
M., et al. (2004). Poplar peroxiredoxin Q. A thioredoxin-linked chloroplast
antioxidant functional in pathogen defense. Plant Physiol. 134, 1027–1038. doi:
10.1104/pp.103.035865
Sarkar, G., and Sommer, S. S. (1990). The megaprimer method of site-directed
mutagenesis. Biotechniques 8, 404–407.
Scheibe, R., and Anderson, L. E. (1981). Dark modulation of NADP-dependent
malate dehydrogenase and glucose-6-phosphate dehydrogenase in the chloroplast. Biochim. Biophys. Acta 636, 58–64. doi: 10.1016/0005-2728(81)90075-X
Stumpp, M. T., Motohashi, K., and Hisabori, T. (1999). Chloroplast thioredoxin
mutants without active-site cysteines facilitate the reduction of the regulatory
disulphide bridge on the g-subunit of chloroplast ATP synthase. Biochem. J. 341,
157–163. doi: 10.1042/0264-6021:3410157
Tarrago, L., Laugier, E., Zaffagnini, M., Marchand, C., Le Marechal, P., Rouhier,
N., et al. (2009). Regeneration mechanisms of Arabidopsis thaliana methionine
sulfoxide reductases B by glutaredoxins and thioredoxins. J. Biol. Chem. 284,
18963–18971. doi: 10.1074/jbc.M109.015487
Yamazaki, D., Motohashi, K., Kasama, T., Hara, Y., and Hisabori, T. (2004). Target
proteins of the cytosolic thioredoxins in Arabidopsis thaliana. Plant Cell Physiol.
45, 18–27. doi: 10.1093/pcp/pch019
Yano, H., Wong, J. H., Lee, Y. M., Cho, M. J., and Buchanan, B. B. (2001). A strategy
for the identification of proteins targeted by thioredoxin. Proc. Natl. Acad. Sci.
U.S.A. 98, 4794–4799. doi: 10.1073/pnas.071041998
Conflict of Interest Statement: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Received: 11 July 2013; accepted: 28 November 2013; published online: 18 December
2013.
Citation: Hara S and Hisabori T (2013) Kinetic analysis of the interactions between
plant thioredoxin and target proteins. Front. Plant Sci. 4:508. doi: 10.3389/fpls.
2013.00508
This article was submitted to Plant Physiology, a section of the journal Frontiers in
Plant Science.
Copyright © 2013 Hara and Hisabori. This is an open-access article distributed under
the terms of the Creative Commons Attribution License (CC BY). The use, distribution
or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance
with accepted academic practice. No use, distribution or reproduction is permitted
which does not comply with these terms.
December 2013 | Volume 4 | Article 508 | 8